1. Field of the Invention
This invention relates to the field of specific binding assays and in particular to assays involving the separation of free and bound species. The ability to measure quantitatively or to identify a wide variety of physiologically active compounds, both naturally occurring and synthetic, has become of increasing importance, both as an adjunct to diagnosis, drug discovery and therapy. For the most part diagnostic assays of physiological fluids or biological samples for one or more analytes have required clinical laboratory determinations although there has been an increasing focus on being able to carry out assay determinations in the doctor's office and in the home. Numerous systems have been developed in efforts to try to address the various problems associated with analyses carried out in the clinical laboratory.
One problem arises with analytes whose presence in biological samples is relatively low. For these analytes greater assay sensitivity is necessary and, consequently, there is a continuing interest in providing improved and alternative methods to those which are presently generally available.
Numerous labels are used in specific binding assays including, for example, enzymes. Enzyme specific binding assays comprise qualitative and quantitative procedures in which a specific binding reaction such as, in immunological cases, an antigen-antibody reaction, is monitored by enzyme activity measurements. The term ELISA is generally used for enzyme immunoassays (EIA) that require a separation step. Reagent excess assays of specific antibodies or antigens that use an enzyme label are sometimes called immunoenzymometric assays. There are two basic types of EIA's: heterogeneous (separation required) and homogeneous (separation free) assays. In the heterogeneous systems, the activity of the enzyme label is not affected by the antigen-antibody reaction and it must be separated into fractions, one being enzyme reagent bound to antibody (or a complex) and the other being free, unbound enzyme reagent, i.e., free and bound species. The enzyme activity of either of these two fractions can be measured.
In the homogeneous systems, the enzyme activity of the assay solution is measured without a prior separation of the antibody-bound enzyme label from the free, unbound one, primarily because the activity of the bound enzyme label is significantly different from the unbound one. The various heterogeneous and homogeneous EIA's can be further characterized as either competitive or non-competitive (immunoenzymometric) assays. The characterization depends on whether the unlabeled antigen and the antigen linked to an enzyme compete for a limited number of antibody binding sites, or whether the antigen or antibody to be measured is allowed to react alone with an excess of immune reactant. For a more detailed discussion of various enzyme assay techniques, see "Enzyme Immunoassay" by Edward T. Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,996,345 and 4,098,876, which listing is not intended to be exhaustive.
Sandwich assays, particularly sandwich immunoassays, are one form of non-competitive assay that has been employed to achieve higher sensitivity in relation to competitive immunoassays. Immunoassays generally involve the use of antibodies, both monoclonal and polyclonal. However, antibodies, even monoclonal antibodies, are relatively inhomogeneous. Additionally, it is difficult to attach an exact number of labels to an antibody all at exactly the same sites. Accordingly, labeling of antibodies tends to further increase heterogeneity of antibody reagents. Other types of binding substances used in binding assays can be even more heterogeneous. For example receptors, particularly membrane bound receptors are frequently isolatable only when bound to other highly heterogeneous components.
Electrophoresis has been used for the separation and analysis of mixtures. Electrophoresis involves the migration and separation of molecules in an electric field based on differences in mobility. Various forms of electrophoresis are known including free zone electrophoresis, gel electrophoresis, isoelectric focusing and isotachophoresis. One approach to immunoassays employs capillary electrophoresis for separation of free and bound label. In capillary electrophoresis electroseparation is performed in tubes or channels of micrometer cross-sectional dimensions. Capillary electrophoresis may be used to separate an antibody-antigen complex from either the unbound form of the antigen or the antibody. Either the bound or free species may be analyzed and quantitated.
Although sandwich specific binding assays can provide much higher sensitivity than competitive assays, the heterogeneity of labeled receptors and antibodies makes the capillary electroseparation difficult to carry out. This results because the unbound and complexed form of the receptor migrates non-uniformly, thus producing broad, poorly defined, rather than sharp, well defined, distributions upon electroseparation analysis. Thus, conventional electroseparation methods may not offer significant advantages for specific binding assay applications.
Various approaches have been disclosed to overcome the inhomogeneity of large biomolecules. In one approach the electrophoretic mobility of a labeled antibody is tailored by attaching charged groups to the same labeled molecule. In another approach one antibody is labeled and the other is highly charged by means of a charge modifying moiety attached to the antibody.
2. Previous Disclosures
Compositions, methods and apparatus for ultrafast electroseparation analysis are described in U.S. Pat. No. 5,630,924 (Fuchs, et al., 1997). See also corresponding PCT application WO 96/33412.
Chen, et al., Clinical Chemistry (1994) 40:1819-1822, describe a method for simultaneous quantification of multiple drug analytes in urine, based on combining immunochemical binding with capillary electrophoretic separation and laser-induced fluorescence.
A rapid hypersensitive flowthrough immunodetection system is disclosed in PCT application WO 93/20449 (Afeyan, et al., 1993).
Greenwood, et al., (Clinical Chemistry (1977) 23(10):1868-1872) describe a radioimmunoassay for digoxin with a fully automated continuous-flow system.
U.S. Pat. No. 4,141,687 (Forrest, et al.) discloses an automatic apparatus and method for the assay of fluid samples.
MacCrindle, et al., (Clinical Chemistry (1985) 31(9):1487-1490) describe a particle concentration fluorescence immunoassay technique for quantification of human immunoglobulins in serum.
A method of solid phase immunoassay incorporating a luminescent label is discussed in U.S. Pat. No. 4,652,533 (Jolley).
Ismail, et al., (Clinical Chemistry (1978) 34(4):571-579) describe the "Southmead System," a simple, fully automated continuous flow system for immunoassays.